The objectives of this proposal are to understand the functional mechanisms of poxvirus transcription activator proteins. For the protypal poxvirus vaccinia, gene expression is regulated primarily at the level of transcription initiation. Most viral genes can be divided into the early or late classes which are activated by class-specific proteins that direct the viral RNA polymerase to different transcription promoters. A major focus of this proposal is the vaccinia early transcription factor (VETF). VETF is a unique promoter binding transcription activator that hydrolyzes adenosine triphosphate for an essential but undefined step in the initiation of RNA synthesis. A series of mechanistic studies are proposed to define the precise step in the initiation of transcription requiring nucleotide hydrolysis. A recently developed series of VETF mutants defective for nucleotide hydrolysis will be characterized for their ability to support early events in the transcription initiation process. In addition a recently discovered factor that enhances VETF promoter binding will be purified and characterized. In order to understand the function of this complex activator protein, the functional domains in VETF responsible for DNA binding, dimerization, and transcription activation will be identified by photoaffinity labeling and mutational approaches. Finally, an investigation into the functional roles for the vaccinia late transcription factors will be initiated. Assays for reconstitution of transcription in vitro will be used to purify previously undefined factors. Recombinant factors will be characterized as to individual roles in the assembly of the late transcription initiation complex. Transcription activator competition experiments will be used to investigate the mechanism of promoter switching that occurs during the course of the infectious cycle. The results of these studies should shed light on our understanding of poxvirus gene regulation and provide insights into mechanisms of transcription initiation in other systems.